Essential Oil Composition of Seven Bulgarian Hypericum Species and Its Potential as a Biopesticide

Hypericum species and especially H. perforatum L. are well known for their therapeutic applications. The present study assessed the essential oil (EO) composition, and antifungal and aphid suppression activity of seven Bulgarian Hypericum species. The EOs were analyzed by GC–MS–FID. Two experiments were conducted. In the first experiment, H. perforatum, H. maculatum, and H. hirsutum were used. Additionally, the EO composition of H. perforatum extracted via hydrodistillation (ClevA) and via commercial steam distillation (Com) were compared. The second experiment compared the EOs of H. perforatum, H. cerastoides, H. rumeliacum, H. montbretii, and H. calycinum (flowers and leaves) extracted via hydrodistillation and collected with n-hexane. Overall, the EO constituents belonged to four classes, namely alkanes, monoterpenes, sesquiterpenes, and fatty acids. The main class for compounds in H. maculatum and H. perforatum (section Hypericum) were sesquiterpenes for both experiments except for H. perforatum (Com). Hypericum montbretii (section Drosocarpium) EO had monoterpenes (38.09%) and sesquiterpenes (37.09%) as major groups, while H. hirsutum EO (section Taeniocarpium) contained predominately alkanes (67.19%). Hypericum hirsutum EO contained cedrol (5.04%), found for the first time in Hypericum species. Fatty acids were the main compounds in H. cerastoides (section Campylopus), while monoterpenes were the most abundant class in H. rumeliacum and H. calycinum EOs. α-Pinene and germacrene D were the major EO constituents of all analyzed Hypericum species except for H. hirsutum and H. cerastoides. Hypericum perforatum EO (Com) had significant repellent and insecticidal activity against two aphid species, Rhopalosiphum padi (Bird Cherry-oat aphid) and Sitobion avenae (English grain aphid) at concentrations of 0%, 1%, 2.5%, 3.5%, 4.5%, and 5%. The tested EOs did not show significant activity against selected economically important agricultural fungal pathogens Fusarium spp., Botrytis cinerea, Colletotrichum spp., Rhizoctonia solani, and Aspergillus sp. The EO of the Hypericum species found in the Bulgarian flora could be utilized for the development of new biopesticides for aphid control.


Results and Discussion
As described in the Materials and Methods section, two independent experiments were performed in this study. The results for the EO composition of the studied Hypericum species were obtained in two different years, with different methodological setups, which is the reason why we do not compare them.

Essential Oil (EO) Compositions for the First Experiment
The EOs of H. perforatum, H. maculatum, and H. hirsutum by ClevA were extracted and analyzed in the first experiment. Analysis of variance (ANOVA) revealed a significant effect of species on the concentration of constituents, and further multiple means comparison results that show which of the species have statistically significantly different mean concentrations are presented in Tables 2 and 3. The EO of H. perforatum by ClevA was compared with the EO of H. perforatum obtained via commercial (Com) steam distillation from Bulgaria (Com, Bul) and USA (Com, USA).  Table 3. Mean concentrations (%) of 2-methyloctane, nonane, α-pinene, 3-methylnonane, β-pinene, cis-β-ocimene, trans-β-ocimene, undecane, caryophyllene oxide, and β-caryophyllene obtained from the six species.

2-Methyloctane
Nonane α-Pinene The compositions of the EOs isolated from Hypericum species of the first experiment are presented in Tables 2 and 3, and Supplementary Tables S1-S3. Gas chromatography (GC) analyses identified between 26 and 50 constituents of H. perforatum, H. maculatum, H. hirsutum, and H. montbretii EOs (Supplementary Tables S1-S4). Overall, the EO constituents belonged to three classes, namely alkanes, monoterpenes, and sesquiterpenes ( Table 2). This study found significant variability in the EO composition of the targeted Hypericum species. The main class of compounds of species from the section Hypericum (H. maculatum, H. perforatum) was the sesquiterpenes, with 66.37% for H. maculatum and 48.08% for H. perforatum, respectively. According to our results and previous research [7,28,37], the sesquiterpenes class was the predominant one for the section Hypericum. However, the results reported for eleven Albanian populations of H. perforatum were different from our results [36]. The latter authors found predominantly sesquiterpenes in six of the populations and predominantly monoterpenes in the other five populations of H. perforatum [36]. Apparently, there was great variation in H. perforatum EO composition and consequently, in the major classes of compounds [7,16,30,31,36,40].
The EO of H. perforatum from this study (the first experiment) is a new chemical type of the species, containing 2-methyloctane, caryophyllene oxide, α-pinene, germacrene D, and β-pinene, respectively (Tables 2 and 3). The composition of H. perforatum EO has been shown to be influenced by a number of factors such as the season, the location, the phenological stage as well as hybridization [18,21]. Due to its easy hybridization, there are many hybrids of H. perforatum with diploid, pentaploid, and triploid forms [9], which may also contribute to the observed diversity in its EO composition.
Generally, the chemical profiles of H. hirsutum, H. montbretii, and H. perforatum in this study were quite different, which contradicts the conclusion of Smelcerović et al. [17]. The cited authors found similarities in the contents of non-terpenes and sesquiterpenes in the EO between the sections Hypericum and Taeniocarpium [17].
Overall, the present study revealed that H. perforatum, H. maculatum, and H. hirsutum, EOs were significantly different with respect to the EO compositions. Secondary metabolites in plants have been successfully used as biomarkers in taxons [14,48]. There have been several published reports in which the authors looked for relationships between EO components as chemotaxonomic markers in the genus Hypericum [17,37,45,49]. However, because genetic and environmental conditions were the main factors that determined the composition of Hypericum EO, its components were deemed insufficient chemotaxonomical markers [17,48].

The Comparison of H. perforatum EO by ClevA and the EO of H. perforatum Obtained via Commercial (Com) Steam Distillation
Hypericum perforatum has been traditionally used in herbal medicines for the treatment of various human disorders such as mild to moderate depression, wound healing, headaches, burns, puncture wounds, vertigo, and others. Generally, Hypericum spp. have low EO yield [42]; however, the EO of the Hypericum species has high value and is much sought after because of its strong antioxidant and antimicrobial activity [50]. Various EO extraction methods such as hydrodistillation, solvent extraction, and critical fluid extraction, have been used; however, steam distillation has been by far the most frequently used method by commercial producers [51]. According to literature data [51], chemical analysis of EOs obtained by different distillation methods revealed roughly the same compounds but in widely different percentages [51]. In this study, the EOs of H. perforatum (Com) from the USA and Bulgaria were compared with the EO of H. perforatum (Clev). The composition of EOs (Com) from the USA and Bulgaria were different and the results are presented in Tables 2 and 3, and Supplementary Table S5. The alkanes were the prevailing chemical group of the EO from the USA (73.41%), while the EO from Bulgaria had monoterpenes and sesquiterpenes as the major group, at similar concentrations of 37.70% and 37.59%, respectively. This result is quite different from the EO profile obtained by the Clevenger apparatus. As mentioned above, 2-methyloctane (10.88%), caryophyllene oxide (15.99%), α-pinene (10.61%), followed by germacrene D (5.44%) and β-pinene (5.95%) were the predominant compounds in the EO obtained by hydrodistillation (Clev) in H. perforatum. 2-Methyloctane (40.89%), nonane (8.80%), α-pinene (13.75%), and 3-methylnonane (11.34%) were the main compounds in the commercial EO from USA (Com), while neryl acetate (9.2%), 2-methyloctane (9.13%), and α-pinene (8.70%) were the main compounds of the commercial EO from Bulgaria (Com) (Supplementary Table S5). The differences may be due to several factors, namely genetic and environmental conditions, and the very similar morphological characteristics of Hypericum species that might play a role in the identification and collection of some other Hypericum species alongside the H. perforatum. Due to the easy hybridization, H. perforatum is known to form hybrids with several other Hypericum species, and these hybrids have many transitional morphological forms [9,52]. Because of the transient forms of H. perforatum, herb pickers may not always precisely distinguish and collect H. perforatum. This may be a possible explanation for the observed differences in EO composition between the USA and Bulgaria (Com).
Hypericum calycinum is a Tertiary relict species native to Southeastern Bulgaria, present in Strandja Nature Park as undergrowth in thermophilic oak forests [55]. According to the Red Book of Bulgaria [55] and The Bulgarian Biodiversity Act [56], the species is endangered (EN B1ab(i,ii) + 2ab(i,ii); C2a(ii)) and protected [55,56]. The species was locally naturalized in Eastern Europe and Eastern Asia [1] and it is widespread and cultivated as an ornamental plant, including in North America. Phytochemical investigations of H. calycinum have been focused mainly on flavonoids, flavonoid glycosides, hyperforin, and cyclohexadienone derivatives [57][58][59]. Previous research on EO content in this species is scarce. Phytochemical investigation of H. calycinum EO in this study showed slight differences in EO composition from flowers and leaves, and the differences were mostly quantitative (Table 4). Generally, the predominant class of the EOs was the monoterpenes, with 73.65% in the flower EO and 54.66% in the leaf EO. β-Pinene, α-pinene, D-limonene, and germacrene D were the prevailing compounds in flowers and leaves, and among them, β-pinene was the most abundant (Table 4, Supplementary Table S6). It can also be noted that in the EO of flowers, n-nonane (5.33%) and β-myrcene (6.48%) were in greater quantity than in the EO of leaves, while α-humulene (6.70%), α-muurolol (torreyol) (5.66%), α-muurolene (3.13%), and β-caryophyllene (4.15%) were characteristic of leaf EO (Supplementary Table S6). There are two literature reports on H. calycinum EO [19,41]. The main components of the EO of the herbarium specimen of this species were α-pinene (24%) and β-pinene (14%) [41], while α-pinene (6.6%), β-pinene (29.2%), limonene (7.2%), β-caryophyllene (3.2%), α-humulene (7.0%), and α-terpineol (11.5%) were the prevailing compounds in aerial shoots of H. calycinum [19].  Tables 5 and 6.  Aphids are economically important pests on agricultural crops and their control is difficult because (1) they reproduce particularly by parthenogenesis and by an amphisexual generation, and (2) they can easily develop resistance to insecticides. In conventional agriculture, the main method for aphid control is the use of chemical pesticides [60]. Since aphids easily develop resistance to chemical pesticides, it is sometimes necessary to increase the applied doses. However, excessive use and high doses of pesticides for aphid control are two of the reasons for the negative effects that pesticides have on human health and the environment [61]. The EOs are volatile compounds, and not only do they repel insects but also have contact and fumigant insecticidal activities, and they can affect insect pests through complex mechanisms [60,61]. Results from this study demonstrated that H. perforatum (Com, Bul) EO application at concentrations of 5%, 4.5%, and 3.5% had a strong insecticidal effect (100%) on both types of aphids within 24th h.

The Pesticide
The application of H. perforatum EO at concentrations of 5% and 4.5% not only killed S. avenae and Rh. padi but also exhibited phytotoxicity on Hordeum vulgare leaves (Table 5). Regarding observed phytotoxicity, H. perforatum EO caused necrotic injuries on the leaves, as most EOs are phytotoxic [61]. At a concentration of 2.5%, the EO showed a lower efficacy for both types of aphids, 83% for S. avenae and 80.9% for Rh. padi, at the 24th h. With increasing duration of treatment (72th h), the insecticidal activity reached 100% (Table 5). Similar effectiveness of extracts from three Hypericum species (H. heterophyllum, H. perforatum, and H. scabrum) was reported by Yaman andŞimşek [62]. The cited authors found that the effectiveness of the extracts of the three Hypericum species was statistically significant depending on the duration of the exposure [62]. After 72th h exposure, mortality ranged from 4.3 to 94.1% for Rhyzopertha dominica, Tribolium confusum, and Sitophilus oryzae, respectively [62]. Moreover, these authors reported that leaf extracts of H. perforatum were more effective on R. dominica, while flower and stem extracts of H. scabrum showed a high toxicity effect on T. confusum and S. oryzae [62].
In general, the insecticidal activity of H. perforatum EO (Com, Bul) at concentrations of 1.5% and 1% was low, and it increased with increasing concentrations of the EO (Table 5). Our results are in agreement with the conclusions of Ba and the environment [61]. The EOs are volatile compounds, and not only do they repel insects but also have contact and fumigant insecticidal activities, and they can affect insect pests through complex mechanisms [60,61]. Results from this study demonstrated that H. perforatum (Com, Bul) EO application at concentrations of 5%, 4.5%, and 3.5% had a strong insecticidal effect (100%) on both types of aphids within 24 th h.
The application of H. perforatum EO at concentrations of 5% and 4.5% not only killed S. avenae and Rh. padi but also exhibited phytotoxicity on Hordeum vulgare leaves (Table 5). Regarding observed phytotoxicity, H. perforatum EO caused necrotic injuries on the leaves, as most EOs are phytotoxic [61]. At a concentration of 2.5%, the EO showed a lower efficacy for both types of aphids, 83% for S. avenae and 80.9% for Rh. padi, at the 24th h. With increasing duration of treatment (72 th h), the insecticidal activity reached 100% (Table 5). Similar effectiveness of extracts from three Hypericum species (H. heterophyllum, H. perforatum, and H. scabrum) was reported by Yaman and Şimşek [62]. The cited authors found that the effectiveness of the extracts of the three Hypericum species was statistically significant depending on the duration of the exposure [62]. After 72 th h exposure, mortality ranged from 4.3 to 94.1% for Rhyzopertha dominica, Tribolium confusum, and Sitophilus oryzae, respectively [62]. Moreover, these authors reported that leaf extracts of H. perforatum were more effective on R. dominica, while flower and stem extracts of H. scabrum showed a high toxicity effect on T. confusum and S. oryzae [62].  In general, the insecticidal activity of H. perforatum EO (Com, Bul) at concentrations of 1.5% and 1% was low, and it increased with increasing concentrations of the EO (Table  5). Our results are in agreement with the conclusions of Baȿ et al. [63] and Parchin and Ebadollahi [64], who found that, with increasing concentrations, H. perforatum EO increased the mortality of Tenebrio molitor L. (Coleoptera: Tenebrionidae) and Tribolium castaneum (Herbst) [63,64]. We should note that the compositions of H. perforatum found in the cited studies [62][63][64] were very dissimilar. For example, Parchin and Ebadollahi [64] reported the dominance of n-decane (59.58%), dodecane (12.93%), ethylcyclohexane (6.84%), 5-methylnonane (4.71%), 3-methylnonane (4.32%), and tetradecane (3.82%) in the H. perforatum EO obtained via steam distillation, while α-pinene (51.2%), 3-carene (7.3%), and α-caryophyllene (5.2%) were the main compounds of the EO in the study of Baȿ et al. [63] obtained via hydrodistillation. In our study, 2-methyloctane, α-pinene, β-himachalene, and neryl acetate were the predominant EO components of H. perforatum (Com, Bul) (Supplementary Table S5). Apparently, the interaction between the components in the EO of H. perforatum exhibits a strong insecticidal effect. Since Hypericum EO exhibits a strong insecticidal effect against Rh. padi (Bird Cherry-oat aphid) and S. avenae (English grain aphid), this EO has the potential to replace harmful chemical insecticides for aphid control.

EO Concentrations (%) After 24 h in % ± SD After 72 h in % ± SD
Repellent Activity of the Commercial EOs of H. perforatum from Bulgaria (Com, Bul) et al. [63] and Parchin and Ebadollahi [64], who found that, with increasing concentrations, H. perforatum EO increased the mortality of Tenebrio molitor L. (Coleoptera: Tenebrionidae) and Tribolium castaneum (Herbst) [63,64]. We should note that the compositions of H. perforatum found in the cited studies [62][63][64] were very dissimilar. For example, Parchin and Ebadollahi [64] reported the dominance of n-decane (59.58%), dodecane (12.93%), ethylcyclohexane (6.84%), 5-methylnonane (4.71%), 3-methylnonane (4.32%), and tetradecane (3.82%) in the H. perforatum EO obtained via steam distillation, while α-pinene (51.2%), 3-carene (7.3%), and α-caryophyllene (5.2%) were the main compounds of the EO in the study of Ba Plants 2023, 12, x FOR PEER REVIEW and the environment [61]. The EOs are volatile compounds, and not only do t insects but also have contact and fumigant insecticidal activities, and they can af pests through complex mechanisms [60,61]. Results from this study demonstrat perforatum (Com, Bul) EO application at concentrations of 5%, 4.5%, and 3.5% ha insecticidal effect (100%) on both types of aphids within 24 th h. The application of H. perforatum EO at concentrations of 5% and 4.5% not o S. avenae and Rh. padi but also exhibited phytotoxicity on Hordeum vulgare leaves Regarding observed phytotoxicity, H. perforatum EO caused necrotic injuries on t as most EOs are phytotoxic [61]. At a concentration of 2.5%, the EO showed a l cacy for both types of aphids, 83% for S. avenae and 80.9% for Rh. padi, at the 24t increasing duration of treatment (72 th h), the insecticidal activity reached 100% Similar effectiveness of extracts from three Hypericum species (H. heterophyllum ratum, and H. scabrum) was reported by Yaman and Şimşek [62]. The cited auth that the effectiveness of the extracts of the three Hypericum species was statistica icant depending on the duration of the exposure [62]. After 72 th h exposure, ranged from 4.3 to 94.1% for Rhyzopertha dominica, Tribolium confusum, and Sitop zae, respectively [62]. Moreover, these authors reported that leaf extracts of H. p were more effective on R. dominica, while flower and stem extracts of H. scabrum a high toxicity effect on T. confusum and S. oryzae [62].  In general, the insecticidal activity of H. perforatum EO (Com, Bul) at conce of 1.5% and 1% was low, and it increased with increasing concentrations of the E 5). Our results are in agreement with the conclusions of Baȿ et al. [63] and Pa Ebadollahi [64], who found that, with increasing concentrations, H. perforatu creased the mortality of Tenebrio molitor L. (Coleoptera: Tenebrionidae) and Trib taneum (Herbst) [63,64]. We should note that the compositions of H. perforatum the cited studies [62][63][64] were very dissimilar. For example, Parchin and Ebado reported the dominance of n-decane (59.58%), dodecane (12.93%), ethylcyc (6.84%), 5-methylnonane (4.71%), 3-methylnonane (4.32%), and tetradecane (3.8 H. perforatum EO obtained via steam distillation, while α-pinene (51.2%), 3-care and α-caryophyllene (5.2%) were the main compounds of the EO in the study of [63] obtained via hydrodistillation. In our study, 2-methyloctane, α-pinene, βlene, and neryl acetate were the predominant EO components of H. perforatum (C (Supplementary Table S5). Apparently, the interaction between the components of H. perforatum exhibits a strong insecticidal effect. Since Hypericum EO exhibit insecticidal effect against Rh. padi (Bird Cherry-oat aphid) and S. avenae (Eng aphid), this EO has the potential to replace harmful chemical insecticides for a trol.

After 24 h in % ± SD
Repellent Activity of the Commercial EOs of H. perforatum from Bulgaria (Com, et al. [63] obtained via hydrodistillation. In our study, 2-methyloctane, α-pinene, β-himachalene, and neryl acetate were the predominant EO components of H. perforatum (Com, Bul) (Supplementary Table S5). Apparently, the interaction between the components in the EO of H. perforatum exhibits a strong insecticidal effect. Since Hypericum EO exhibits a strong insecticidal effect against Rh. padi (Bird Cherry-oat aphid) and S. avenae (English grain aphid), this EO has the potential to replace harmful chemical insecticides for aphid control.
Repellent Activity of the Commercial EOs of H. perforatum from Bulgaria (Com, Bul) The repellent activity of H. perforatum EO was evaluated at 6 concentrations: 0%, 1%, 2.5%, 3.5%, 4.5%, and 5% on Rh. padi (Bird Cherry-oat aphid) and on S. avenae (English grain aphid) ( Table 6). The highest repellent activity was observed with the EO applications at 5% and 4.5% for both types of aphids (Table 6). Similar results were found for Juniperus sabina EO (Male, Female) and J. communis L., J. oxycedrus L., J. pygmaea C. Koch., and J. sibirica Burgsd, where a 4.5% concentration rate had a stronger repellant effect on the S. avenae aphids than on the Rh. padi aphids [65,66]. A literature review yielded no studies on the repellent effect of H. perforatum EO. The repellent activity of the EO from aniseed (Pimpinella anisum L.), peppermint (Mentha piperita L.), and lemongrass (Cymbopogon flexuosus (Nees ex Steud.) W. Watson) against Rh. padi has been reported by Pascual-Villalobos et al. [67], who found that some EO constituents were active: carvone increased mobility, whilst cis-jasmone repelled Rh. padi at a very low dose (0.02 µL/cm 2 of the treated leaf) [ (Tables 7 and 8). The antifungal activity of the H. perforatum EO on the mycelial growth of the tested pathogens was variable on different days after the treatment. On the third day of the experiment, we observed higher growth of fungal colonies of Fusarium sp. (104% at 1 µL mL −1 and 125.5% at 2 µL mL −1 ), B. cinerea (108.54% at 1 µL mL −1 and 122.25% at 2 µL mL −1 ), and Colletotrichum sp. (101.3% at 1 µL mL −1 and 102% at 2 µL mL −1 ) in treated variants compared to the control. The high inhibition of the mycelial growth of Aspergillus sp. by the EO was seen only on the third day and decreased during the experiment. The diameter of the fungal colonies, except Colletotrichum sp., became equal on the ninth day in all tested variants. Table 7. Inhibitory effect of commercial Hypericum perforatum essential oils (Com, Bul) on plant pathogenic fungi (mean ± SD).

Control
It is likely that the more substantial initial growth of Fusarium sp. and B. cinerea was due to some of the EO components of H. perforatum that have a stimulatory effect on these phytopathogens. It was previously reported that the H. hyssopifolium and H. heterophyllum EOs increased the growth of some fungal species [3]. The inhibitory effect of EOs on pathogenic fungi depends on their application rate and the duration of the inhibition period [68]. Nosrati et al. [69] reported that samples treated with 1 µL of spearmint (Mentha spicata L.) EO showed a slow decrease in the antifungal activity against Fusarium oxysporum f. sp. radicis-cucumerinum throughout the incubation period. Table 8. Inhibitory effect of commercial Hypericum perforatum essential oils on plant pathogenic fungi (mean ± SD). Both β-caryophyllene oxide and α-terpineol were identified as constituents of the Hypericum species EO and have been previously reported as mycelial growth inhibitors against fungi [3]. High values of the inhibitory effect of the Ocimum sanctum L. EO against target filamentous fungi may be due to the characteristically high content of the monoterpenoid alcohol linalool and the phenylpropanoid estragole. It can be noted that eugenol, linalool, and thymol were among the plant constituents that have significant antifungal activity [70].

Plant Materials
Plant materials of H. perforatum L., H. maculatum Crantz., H. hirsutum L. were collected in 2019 for the first experiment (Table 9) (Table 9). The samples of H. calycinum were collected ex situ from the Experimental and Teaching Garden of the Agricultural University, Plovdiv, Bulgaria. Voucher specimens of all these species were deposited at the Herbarium of the Agricultural University, Plovdiv, Bulgaria (SOA).

Essential Oil (EO) Extraction from the Hypericum Biomass Samples
Two separate experiments were conducted: (i) in the 2019 and (ii) 2020 cropping seasons. The two experiments were independent, so we did not compare the results between the different collection years.

First Experiment
In the first experiment, conducted in 2019, the plant materials of H. perforatum, H. maculatum, and H. hirsutum were collected in the flowering stage. The locations, coordinates, and altitude are shown in Table 9. The EOs were analyzed following extraction via hydrodistillation. The samples of the four species were dried in laboratory conditions, in a shady location. The 100 g samples (inflorescences with a small part of the stem) of each four Hypericum species were cut into small pieces and put in 2 L distillation Clevenger units (ClevA) (Laborbio Ltd. Sofia, Bulgaria, www.laborbio.com, accessed on Feb 8th, 2023). We used 800 mL of water, resulting in a 1:8 ratio of plant material to water. The

Second Experiment
In the second experiment conducted in 2020, H. cerastoides, H. rumeliacum, H. montbretii, and H. calycinum (flower) and H. calycinum (leaves) samples were collected. The exact weight of the fresh materials of the target Hypericum species is shown in Table 9. The EOs of Hypericum species in the second experiment were extracted by hydrodistillation at the University of Food Technologies in Plovdiv. The EOs were extracted by hydrodistillation for 2 h 30 min in a modified Clevenger-type glass apparatus. Because of the low EO yield and difficulty with the oil collection, n-hexane was used to wash the sides of the apparatus and collect all the oil. Therefore, the EOs in the second experiment were dissolved in n-hexane.

Gas Chromatography (GC)-Mass Spectrometry (MS) Analyses of the EOs The First Experiment-Gas Chromatography-Mass Spectrometry-Flame Ionization Detection (GC-MS-FID) Essential Oil Analysis
GC-MS-FID analysis of Hypericum hirsutum, Hypericum maculatum, and Hypericum perforatum samples and Hypericum perforatum standard EOs from the first experiment was performed by placing 50 µL of oil (weight also recorded) into a 10 mL volumetric flask. Samples were brought to volume with chloroform.
Oil samples were analyzed by GC-MS-FID on an Agilent (Santa Clara, CA, USA) 7890A GC system coupled to an Agilent 5975C inert XL MSD. Chemical standards and oils were analyzed using a DB-5 column (30 m × 0.25 mm fused silica capillary column, film thickness of 0.25 µm) operated using an injector temperature of 240 • C, column temperature of 60 to 240 • C at 3 • C/min and held at 240 • C for 5 min, helium as the carrier gas, an injection volume of 1 µL (split ratio 25:1), and an MS mass range from 50 to 600. The FID temperature was 300 • C. Post-column splitting was performed so that 50% of outlet flow proceeded to FID and 50% to mass spectrometry (MS) detection.
Compounds were identified by Kovats Index analyses and comparison of mass spectra with those reported in the Adams and NIST mass spectra databases as well as a direct comparison of MS and retention time to authentic standards. Commercial standards of nonane, decane, 2-nonanone, undecane, decanal, α-longipinene, trans-caryophyllene, transβ-farnesene, (+)-valencene, caryophyllene oxide, cedrol, sabinene, β-pinene, myrcene, pcymene, nonanal, terpinen-4-ol, isoledene, β-caryophyllene, α-humulene, trans-β-farnesene, (+)-valencene, ledol, and 2-pentylfuran were obtained from Sigma-Aldrich (St. Louis, MO, USA). Germacrene D was obtained from Supelco (via Sigma-Aldrich, St. Louis, MO, USA). Compounds were quantified by performing area percentage calculations based on the total combined FID area. For example, the area for each reported peak was divided by the total integrated area from the FID chromatogram from all reported peaks and multiplied by 100 to arrive at a percentage. The percentage of a peak is a percentage relative to all other constituents integrated into the FID chromatogram.

Second Experiment GC-MS Analysis
The chemical composition of the investigated Hypericum essential oils from the second experiment in two repetitions was determined by GC-MS analysis. Compounds were separated using an Agilent 5890A gas chromatograph coupled with an Agilent 5795C MSD and fitted with a fused silica capillary column HP-5MS (5% phenylmethylpolysiloxane, 30 m × 0.25 mm i.d., 0.25 µm film thicknesses) (Agilent Technologies, Santa Clara, CA, USA). The temperature was programmed from 60 • C to 300 • C with 5 • C/min, held for 10 min; the injection volume was 1.0 µL and the split ratio was 50:1. The flow rate of the He (carrier gas) was 0.8 mL/min. Electron ionization (EI) mass spectra were recorded in the positive ion mode at 70 eV; acquisition mass range was 30-600 m/z. The ion source transfer and the line were set at 250 • C.
GC analysis of the EO volatile components was performed using an Agilent 5890A gas chromatograph equipped with a flame ionization detector (FID) on an Agilent capillary column, HP-5 (30 m × 0.32 mm; film thickness 0.25 µm) (Agilent Technologies). The temperature program conditions were the same as with GC-MS analysis. The temperatures of the detector and the injector were 280 • C and 220 • C, respectively. FID temperature was set at 260 • C. The carrier gas was helium at a flow rate of 1.0 mL/ min 1 .
A mixture of aliphatic hydrocarbons from C6 to C32 (Sigma Aldrich, St. Louis, MO, USA) was injected into the GC system under the above temperature program in order to calculate the retention index (RI) of each compound in the samples and the percentage compositions of the individual components were obtained from electronic integration measurements using FID.
Compound identification was carried out by comparing the retention time, RI, and mass spectra of the chromatographic peaks with those in the commercial NIST'08 (National Institute of Standards and Technology, Gaithersburg, MD, USA) and Adams libraries [71]. The insecticidal activity of H. perforatum EO (Com, Bul) was tested according to a method described in Konstantopoulou et al. [72]. The EO was applied at concentrations of 0% (control), 1%, 2.5%, 3.5%, 4.5%, and 5% in three replicates. Two species of adult wingless forms of aphids, Rhopalosiphum padi (Bird Cherry-oat aphid) and Sitobion avenae (English grain aphid), were used for insecticidal activity, in three replicates. The procedure of evaluating the insecticidal activity of the EO has been described previously [65]. Hypericum perforatum EO was diluted in an aqueous solution with an emulsifier of 0.1% polysorbate 80. The control (0%) was treated with a 0.1% aqueous solution of polysorbate 80. Two microliters of the solution (0%, 1%, 2.5%, 3.5%, 4.5%, and 5%) were applied directly to barley leaves with the aphid colonies. The leaves were then dried on a filter paper and transferred to Petri dishes as described by Konstantopoulou et al. [72]. The Petri dishes were covered with cheesecloth (44 g/m 2 ). The effect of the application (knockdown or mortality) was observed after 24 and 72 h. The results (knockdown and mortality) were compared with controls. The efficacy of EO concentrations was calculated according to the Henderson-Tilton formula [73]:

The Pesticide
where T a -number insects after treatment; T b -number insects before treatment; C b -the number of insects in control before treatment plot; C a -the number of insects in control after treatment plot.
The Repellent Activity of H. perforatum EO (Com, Bul) against Rhopalosiphum padi and Sitobion avenae The repellent activity of the EO of H. perforatum (Com, Bul) was tested at concentrations of 0%, 1%, 2.5%, 3.5%, 4.5%, and 5% in three replicates. Two microliters of EO was tested for repellency by using the Petri dish analysis according to Jiang et al. [74]. The H. perforatum EO (Com, Bul) was diluted with an aqueous solution with an emulsifier, 0.1% polysorbate 80, as described previously [65] with one treated leaf (with different concentration of EO), and one non-treated leaf (control, 0.1% polysorbate 80). The leaves were five cm long and positioned parallel at a distance of 2 cm between them, on a moistened filter paper in Petri dishes [74]. Ten leafless aphids were introduced into each Petri dish between the treated leaf and non-treated leaf (control). The Petri dishes were then covered with a cheesecloth (44 g/m 2 ). The repellent effect was observed and recorded after 24 h. Descriptive statistical analyses of the data were performed by calculating the mean and the standard deviation (SD) values of the three replicates [75] An agar dilution method was used for the preliminary testing of the antifungal activity of H. perforatum, namely on five plant pathogens. The EO was diluted in potato dextrose agar (PDA) at two concentrations (1 µL mL −1 and 2 µL mL −1 ). PDA with EO was poured onto Petri dishes (90 mm/d). Discs (five mm) were cut from the periphery of a 10-day-old culture of tested fungi and aseptically put in the center of the Petri dishes. Pure PDA medium with sterile distilled water (without EO) was used as the control. The inoculated Petri dishes were placed at 22 • C for nine days. All experiments were conducted in four replications. The diameter of the fungal colony was measured on the 3rd, 6th, and 9th day. The percent inhibition of the radial growth of the tested fungi was calculated using the following formula: (DC − DT)/DC × 100% (2) where DC is the diameter of the control colony, and DT is the diameter of the treatment colony. Mean and SD values were also calculated.

Statistical Analyses
For the data obtained from the first experiment, analysis of variance (ANOVA) of a completely randomized design (CRD), also known as one-way ANOVA, was conducted to determine the effect of species (five levels: H. hirsutum, H. maculatum, H. perforatum, H. perforatum (Com, USA), and H. perforatum (Com, Bul), on the concentrations of 15 constituents (2-methyloctane, nonane, α-pinene, 3-methylnonane, β-pinene, (Z)-β-ocimene, (E)-β-ocimene, undecane, caryophyllene oxide, (E)-caryophyllene, (E)-β-farnesene, germacrene D, δ-cadinene, α-epi-cadinol, and α-cadinol), and four classes (monoterpenes, sesquiterpenes, alkanes, and other). The analyses were completed using the GLM Procedure of SAS [76]. Since the effect of species was significant (p-value < 0.05) on the concentrations of all 19 constituents, further multiple means comparison was completed using Tukey's multiple range test at 5% level of significance and letter groupings were generated. For each response variable, the validity of the normal distribution of the error terms assumption was verified by generating a normal probability plot of residuals and testing for normality of the error terms using the residuals, and the validity of the constant variance assumption of the error terms was verified by plotting the residuals vs. the fitted values, as described in Montgomery [75].
For the data from the second experiment, descriptive statistics (mean and standard deviation) were calculated using the three replicates.

Conclusions
Generally, the EOs of the seven Hypericum species from Bulgaria had very different compositions, especially H. perforatum. The testing of EOs of H. hirsutum, H. montbretii, H. cerastoides, H. rumeliacum, and H. calycinum in Bulgarian populations was conducted for the first time. The application of H. perforatum EO (Com) at concentrations of 5% and 4.5% exhibited high repellent activity and was effective against two aphid species: S. avenae and Rh. padi. In our study, H. perforatum EO did not exhibit substantial antifungal activity against R. solani, Fusarium sp., B. cinerea, and Aspergillus sp. but had a moderate inhibitory effect on Colletotrichum sp. Since there is great variability in the compositions of Hypericum EOs, it is necessary to select and grow a specific accession with a desirable composition in order to standardize the EO composition. The standardized EO compositions are considered alternative products with the potential to substitute synthetic pesticides in controlling pests on agricultural crops. These molecules constitute a significant source of biologically active components-antioxidant, antibacterial, insecticidal, fungicidal, and herbicidal. Therefore, EOs have potential as biological products in integrated and ecological plant protection.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants12040923/s1, Table S1: Essential oil composition of Hypericum perforatum; Table S2: Essential oil composition of Hypericum hirsutum; Table S3: Essential oil composition of Hypericum maculatum; Table S4: Essential oil composition of Hypericum montbretii; Table S5: Essential oil composition of Hypericum perforatum, commercial from USA and Bulgaria; Table S6: Essential oil composition of Hypericum perforatum, H. rumeliacum from the second experiment.